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Steroid hormones are vital for a successful pregnancy. The placenta is attached to the uterine wall and is the major organ of communication between the mother and the fetus through the umbilical cord and the transfer of compounds (including the production and actions of steroids) across the villous placenta. Therefore, a correct understanding and measurement of steroid levels across the maternal–placental–fetal interface is essential. We have experience spanning more than two decades and have published more than 40 papers using a variety of methods to assess circulating and placental steroid levels. In this review, we discuss various methods for steroid detection and quantitation, as well as their advantages and disadvantages. This document provides technical guidance for best practices that, in our estimation, can assist researchers in more easily and correctly performing these studies. Critical methodological considerations, including tissue collection, tissue processing, and analytical factors (sensitivity, selectivity, matrix effects, and internal standards), are covered. We highlight important differences between human and rodent tissues as they relate to steroid levels in pregnancy and the interpretation of results, and provide guidance for best practices in future studies.